We focused our research efforts to define the role of inactivation of the retinoblastoma gene (Rb) in the etiology of human cancer and to characterize the mechanisms underlying RB-mediated tumor suppressor pathways. We have identified the Rb gene as the first tumor suppressor gene targeted for mutations in the common adult malignancies, small cell lung cancer (SCLC) and non-SCLC. We extended these observations by demonstrating (l) that 90% of SCLC samples and 15% of non-SCLC had absent or aberrant RB protein expression, (2) that naturally occurring mutations isolated from lung cancer samples had targeted an internal protein binding domain that is essential for RB-mediated tumor suppressor activity, and (3) that reintroduction of the Rb gene into RB(-/-) lung cancer cells was associated with reversion of tumorigenicity. We defined the properties of a unique type of mutant RB gene product that is transmitted in the germline of several different families with incomplete penetrance of familial retinoblastoma. This RB mutant exhibited partial inactivation and has allowed us to propose a hypothesis for a molecular mechanism to explain the phenotype of incomplete penetrance. Finally, the observation that tumor suppression depends on phosphorylation and conformational changes within RB that are tightly synchronized to cell cycle events has suggested several approaches to study the biology of the Rb gene. These include the identification and characterization of cellular proteins that reversibly bind to the hypophosphorylated form of RB, as well as the regulation of the activity of the cyclin dependent kinase (cdk):cyclin enzymes that phosphorylate RB in vivo. We have obtained full length cDNAs, generated specific polyclonal antisera, and have characterized the binding properties of two nuclear proteins designated retinoblastoma binding protein-1 (RBP1) and RBP2. Although the role of these proteins is still unknown, preliminary experiments demonstrate features which suggest they may function as transcription factors. We have examined the protein expression of the cdk4 and cdk6-inhibitor p16(INK)4. Our studies have shown a striking inverse correlation between the expression of wildtype RB and p16(INK)4. These findings confirmed the presence of a common p16(INK):RB growth suppressor pathway in human cancers and provided evidence that the CDKN2 gene is a specific target for mutational events.